METHODS OF CULTURE \ND OF EXAMINATION.^ 107 



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rinsed in water, dried, and mounte^4^ 'Canada >balsa/n. 

 Stained in this way certain bacteria assume a bluish-black 

 color, while others yield up the stain completely on decol- 

 orization. Gram's method, which is of especial importance 

 in the identification of bacteria, is most difficult of applica- 

 tion. The slightest variations in its technic may render the 

 results doubtful. It is well, therefore, to make a control- 

 stain in every instance, examining simultaneously, in 

 addition to the bacteria under investigation, another variety 

 of bacteria that with certainty either stains (staphylococci) 

 or decolorizes (bacterium coli). 



Giinther's Modification of Gram's Method. After 

 exposure to the action of the iodin potassium-iodid solution, 

 the specimen is placed for half a minute in alcohol, then for 

 a short time (ten seconds) in three per cent, hydrochloric- 

 acid alcohol, and, finally, for complete decolorization, in 

 pure alcohol. Before mounting, the sections are cleared in 

 oil of cloves or in xylol. 



Weigert's Modification of Gram's Method. After 

 treatment with aniline-water gentian -violet solution, the 

 specimen is rinsed in water, and the section is spread upon 

 a slide, iodin potassium-iodid is added, and then removed 

 with bibulous paper. Next follow differentiation, dehydra- 

 tion, and clearing with aniline oil and xylol, and mounting 

 in Canada balsam. 



Double Staining. To make a contrast between the 

 bacteria and the surrounding tissue, it is advisable, after 

 staining by Gram's method, to stain the sections further 

 with a dilute watery solution of vesuvin, safranin, or car- 

 mine, and to rinse in alcohol. In this way, however, the 

 bacteria under some circumstances lose a portion of their 

 stain. For this reason it is preferable to apply the contrast- 

 stain before using Gram's method of staining the bacteria. 

 To this end the sections are washed in water, stained in 

 picrocarmine solution, again washed in water and placed in 

 alcohol, after which the staining, according to the method 

 of Gram, can be undertaken at once or at any convenient 

 time. The staining of the tissues is in no way interfered 

 with by Gram's method. Not all bacteria remain stained 

 after application of Gram's method : a very consider- 

 able number can not be demonstrated by this means. 

 Particulars in this regard will be found in the special 

 section. 



